The present invention relates to a process for the preparation of acyl phosphate esters, novel acyl phosphate esters and their use in the preparation of modified proteins.
Acyl phosphates (mixed anhydrides of a carboxylic acid and phosphoric acid) occur as intermediates in many biochemical processes. The acyl phosphates may function as activated carboxylic acids thus promoting the transfer of the acyl group to an acceptor. For example, in the formation of intramitochondrial acyl-coenzyme A in some organisms, acetate is activated by acetate kinase through reaction with ATP to produce acetyl phosphate prior to transfer of the acetyl group to coenzyme A. (Walsh, C. Enzymatic Reaction Mechanisms, W. H. Freeman Co.: New York, 1979 pp. 234-238). Amino acids are activated as aminoacyl adenylates prior to their incorporation into peptides and proteins on ribosomes (Ibid, pp. 241-248).
The procedures reported for preparing acyl phosphate esters have many limitations. Phenyl acetyl phosphate has been prepared from phenyl phosphate and acetic anhydride (Jencks, W. P.; Carriuolo, J., J. Biol. Chem, 1959, 234, 1272, 1280; DiSabato, G., Jencks, W. P., J. Am Chem. Soc. 1961, 83, 4400; Oestreich, C. H., Jones, M. M., Biochemistry 1966, 5, 2926; Oestreich, C. H., Jones, M. M. Biochemistry 1967, 6, 1515; Briggs, P. J. et al, J. Chem. Soc. B. 1970, 1008) but the extension of this method to the use of alkyl phosphate in place of phenyl phosphate gives impure, uncharacterized products (Jencks, W. P. Carriuolo, J., J. Biol. Chem., 1959, 234, 1272). Classical methods for the synthesis of aminoacyladenylates (coupling of . . . the N-protected amino acid with adenylic acid using dicyclohexylcarbodiimide) also gives impure products (Berg, P., J. Biol. Chem. 1958, 233, 608).
Methyl acetyl phosphate has been prepared by reacting dimethyl acetyl phosphate with sodium iodide in acetone. (Kluger, R., Tsui, W. C., J. Org. Chem. 1980, 45, 2723 and Kluger, R., Tsui W. C., Biochem. and Cell Biol. 1986, 64, 434). The synthesis of the dimethyl acetyl phosphate involves refluxing acetyl chloride and trimethyl phosphate for an extended period (Whetstone, R., U.S. Pat. No. 2,648,896; and Chem. Abstr. 1954, 48, 8250; and Kluger, R., Wasserstein, P., Biochemistry 1972, 11, 1544). However, the present inventors have found that this reaction may not be extended to more complex acid chlorides or to diacid chlorides, for example, succinyl chloride or fumaryl chloride. A potential alternative route for the preparation of dimethyl acetyl phosphate is to react an acyl halide with a dimethyl phosphate salt. Acetyl chloride has been reported to react with triethylammonium dimethyl phosphate to produce dimethyl acetyl phosphate but the material was isolated in an "impure and unstable state" (Avison, A.W.D., J. Chem. Soc. 1955, 732). A further limitation of this alternative route is that it must rigorously exclude water in order to be effective, since the diester is very reactive in water (Kluger, R., Wasserstein, P., Biochemistry 172, 11, 1544).
Monoesters of acyl phosphates have been found to be stable in neutral aqueous solutions (Klimman, J. P., Samuel, D. Biochemistry, 1971, 10, 2126) and have been reported to acetylate amino groups in sites which bind anions or proteins (Khger, R., Tsui W. C., J. Org. Chem., 1980, 45, 2723; Khger, R., Tsui W. C., Blochem. and Cell Biol. 1986, 64, 434, and Ueno, H. et al, Arch. Blochem. Biophys. 1986, 244, 795). Kern et al (Biochemistry, 1985, 24, 1321) have shown that aminoacyladenylate selectively acylates amino residues of an aminoacyl t-RNA synthetase when it is produced by the enzyme from an amino acid which is not its normal substrate. Methyl acetyl phosphate hah been found to specifically acetylate amino groups exclusively in the region of the 2-3-diphosphoglycerate binding site in hemoglobin. (Ueno H., et al, Arch. Blochem. Biophys. 1986, 244, 795 and Ueno, H. et al, 1989, 26, 12344).